Gao Yang, You Xinran, Liu Yubo, Gao Fei, Zhang Yuan, Yang Jianrong, Yang Chen
Jiangsu Key Laboratory of Oral Disease, The Affiliated Hospital of Stomatology, Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China.
Department of Oral and Maxillofacial Surgery, The Affiliated Hospital of Stomatology, Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China.
Exp Ther Med. 2020 Mar;19(3):2202-2210. doi: 10.3892/etm.2020.8475. Epub 2020 Jan 28.
The NOD-like receptor protein 3/caspase-1 inflammasome can be activated in human dental pulp tissue and fibroblasts; however, the underlying mechanisms are poorly understood. In the present study, lipopolysaccharide (LPS) was used to treat dental pulp cells to establish an inflammation model. Cell viability was examined by sulforhodamine B assay. Interleukin (IL)-1β, caspase-1, microtubule-associated protein-1 light chain 3-II/I and p62 were determined by western blotting and ELISA. The phosphorylation (p-) levels of NF-κB and NF-κB inhibitor (IκB)α protein were observed by western blotting. The results demonstrated that LPS induced pyroptotic cell death in cultured dental pulp cells, which was supported by the increased levels of IL-1β, IL-18 and caspase-1. Rapamycin and 3-methyladenine (3-MA) were used to activate and inhibit autophagy, and it was observed that LPS increased autophagy and rapamycin reduced LPS-induced dental pulp cell pyroptosis. However, 3-MA aggravated LPS-induced dental pulp cell pyroptosis. In addition, LPS inhibited the expression of IκBα, but increased the expression of p-NF-κB. Compared with the LPS group, 3-MA further inhibited the expression of IκBα but promoted the expression of p-NF-κB. However, rapamycin produced the opposite results to LPS. Under LPS treatment, the NF-κB pathway inhibitor BAY11-7082 further enhanced the inhibitory effects of rapamycin, but inhibited the promoting effects of 3-MA on the protein expression levels of IL-1β and caspase-1. The results of the present study demonstrated that there is an important crosstalk between autophagy, pyroptosis and the NF-κB pathway, and that the modulation of pyroptosis in dental pulp cells may be a promising strategy to pulpitis therapy.
核苷酸结合寡聚化结构域样受体蛋白3/半胱天冬酶-1炎性小体可在人牙髓组织和成纤维细胞中被激活；然而，其潜在机制尚不清楚。在本研究中，使用脂多糖（LPS）处理牙髓细胞以建立炎症模型。通过磺酰罗丹明B测定法检测细胞活力。通过蛋白质印迹法和酶联免疫吸附测定法测定白细胞介素（IL）-1β、半胱天冬酶-1、微管相关蛋白-1轻链3-II/I和p62。通过蛋白质印迹法观察核因子κB（NF-κB）和NF-κB抑制剂（IκB）α蛋白的磷酸化（p-）水平。结果表明，LPS诱导培养的牙髓细胞发生焦亡性细胞死亡，IL-1β、IL-18和半胱天冬酶-1水平升高支持了这一结果。使用雷帕霉素和3-甲基腺嘌呤（3-MA）激活和抑制自噬，观察到LPS增加自噬，雷帕霉素减少LPS诱导的牙髓细胞焦亡。然而，3-MA加剧了LPS诱导的牙髓细胞焦亡。此外，LPS抑制IκBα的表达，但增加p-NF-κB的表达。与LPS组相比，3-MA进一步抑制IκBα的表达，但促进p-NF-κB的表达。然而，雷帕霉素产生了与LPS相反的结果。在LPS处理下，NF-κB途径抑制剂BAY11-7082进一步增强了雷帕霉素的抑制作用，但抑制了3-MA对IL-1β和半胱天冬酶-1蛋白表达水平的促进作用。本研究结果表明，自噬、焦亡和NF-κB途径之间存在重要的相互作用，调节牙髓细胞中的焦亡可能是牙髓炎治疗的一种有前景的策略。
